Phenyloxadiazole derivatives as pgds inhibitors

ABSTRACT

wherein R1, R2 and R3 are as defined herein, a pharmaceutical composition comprising the compound, intermediates and processes for making said compounds, and the use of the compound to treat allergic and/or inflammatory disorders, particularly disorders such as allergic rhinitis, asthma, chronic obstructive pulmonary disease (COPD) and age-related macular degeneration (AMD).

FIELD OF THE INVENTION

The present invention is directed to phenyloxadiazole compounds, theirpreparation, pharmaceutical compositions containing these compounds, andtheir pharmaceutical use in the treatment of disease states capable ofbeing modulated by the inhibition of the prostaglandin D synthase.

BACKGROUND OF THE INVENTION

Allergic rhinitis, the most common atopic disease, has an estimatedprevalence ranging from about 5 to about 22 percent of the general humanpopulation and is characterized by the symptoms of sneezing, nasaldischarge, and nasal congestion. These symptoms are believed to betriggered by multiple mediators released from mast cells and otherinflammatory cells. Current therapies, such as antihistamines, dealeffectively with the sneezing and nasal discharge, but have littleeffect on congestion, which is a key symptom affecting the quality oflife of patients.

Local allergen challenge in patients with allergic rhinitis, bronchialasthma, allergic conjunctivitis and atopic dermatitis has been shown toresult in rapid elevation of prostaglandin D2 “(PGD2)” levels in nasaland bronchial lavage fluids, tears and skin chamber fluids. PGD2 hasmany inflammatory actions, such as increasing vascular permeability inthe conjunctiva and skin, increasing nasal airway resistance, airwaynarrowing and cosinophil infiltration into the conjunctiva and trachea.PGD2 is the major cyclooxygenase product of arachidonic acid producedfrom mast cells on immunological challenge [Lewis, R A, Soter N A,Diamond P T, Austen K F, Oates J A, Roberts L J II, Prostaglandin D2generation after activation of rat and human mast cells with anti-IgE,J. Immunol. 129, 1627-1631, 1982]. Activated mast cells, a major sourceof PGD2, are one of the key players in driving the allergic response inconditions such as asthma, allergic rhinitis, allergic conjunctivitis,allergic dermatitis and other diseases [Brightling C E, Bradding P,Pavord I D, Wardlaw A J, New Insights into the role of the mast cell inasthma, Clin. Exp. Allergy 33, 550-556, 2003].

In the presence of sulfhydryl compounds, PGD2 is formed by theisomerization of PGH2, a common precursor of prostanoids, by catalyticaction of prostaglandin D synthase “(PGDS)”. There are two isoforms ofthe PGDS enzyme: L-PGDS; and H-PGDS. H-PGDS is a cytosolic enzyme, whichis distributed in the peripheral tissues, and which is localized in theantigen-presenting cells, mast cells, megakaryocytes, and Th2lymphocytes. The action of the product PGD2 is mediated by G-proteincoupled receptors: D prostaglandin “(DP)” and crTH2. See (1)Prostaglandin D Synthase: Structure and Function. T. Urade and O.Hayaishi, Vitamin and Hormones, 2000, 58, 89-120, (2) J. J. Murray, N.Engl. J. Med., 1986 Sep. 25; 315(13):800, and (3) Urade et. al, J.Immunology 168: 443-449, 2002.

Without wishing to be bound by theory, inhibiting the formation of PGD2should have an effect on nasal congestion and, therefore, be oftherapeutic benefit in allergic rhinitis. In addition, we believe that aPGDS inhibitor should be of therapeutic benefit in a number of otherindications such as bronchial asthma, age-related macular degeneration(AMD) an/or or chronic obstructive pulmonary disease (COPD).

Age-related macular degeneration (AMD) is a degenerative and progressiveocular disease that results in loss of fine, central vision due to thedegeneration of the macula. AMD is the most common cause of blindness inEurope and the United States for individuals over 50 years of age.

Chronic obstructive pulmonary disease (COPD) is a progressive,inflammatory disease that involves chronic bronchitis and emphysema.Symptoms include airflow limitation, excessive mucus production,coughing, reduced exercise capacity and reduced quality of life.

PGDS inhibitors have been reported. The compound, HQL-79, is reported tobe a weak PGDS inhibitor, and is antiasthmatic in guinea pig and ratmodels (Matsusshita, et al., Jpn. J. Pharamcol. 78: 11, 1998). Thecompound Tranilast is described as a PGDS inhibitor. (Inhibitory Effectof Tranilast on Prostaglandin D Synthesase. K. Ikai, M. Jihara, K.Fujii, and Y. Urade. Biochemical Pharmacology, 1989, 28, 2773-2676). Thefollowing published patent applications also disclose PGDS inhibitors:

US2008/0207651A1 and US2008/0146569A1—pyridine and pyrimidinecarboxamides;JP2007-51121—pyrimidine carboxamides;WO2007/007778—benzimidazole derivatives;WO2008/122787—piperazine(thio)carboxamides; and WO2005/094805—imine andamide derivatives.

SUMMARY OF THE INVENTION

The present invention is directed to a compound of formula (I):

wherein:R1 is hydrogen or C₁-C₆alkyl;R2 is hydrogen, halogen or C₁-C₃alkyl; andR3 is hydroxyalkylor a pharmaceutically acceptable salt thereof.

Another aspect of the present invention is a pharmaceutical compositioncomprising a pharmaceutically effective amount of a compound accordingto formula (I) and a pharmaceutically acceptable carrier.

Another aspect of the present invention is directed to a method oftreating allergic and/or inflammatory disorders, particularly disorderssuch as allergic rhinitis, asthma, chronic obstructive pulmonary disease(COPD) and/or age-related macular degeneration (AMD) in a patient inneed thereof by administering to the patient a compound according toformula (I). Another aspect of the invention is a process for preparingcompounds of formula (I).

DETAILED DESCRIPTION OF THE INVENTION Definition of Terms

As used above, and throughout the description of the invention, thefollowing terms unless otherwise indicated, shall be understood to havethe following meanings:

“Alkyl” means straight or branched aliphatic hydrocarbon having 1 toabout 20 carbon atoms. Particular alkyl has 1 to about 12 carbon atoms.More particular alkyl is lower alkyl. Branched means that one or morelower alkyl groups such as methyl, ethyl or propyl are attached to alinear alkyl chain. “Lower alkyl” means 1 to about 4 carbon atoms in alinear alkyl chain that may be straight or branched.

“Hydroxyalkyl” means OH-alkyl-. Particular hydroxyalkyl ishydroxy(C₁-C₆)alkyl-. Exemplary hydroxyalkyl includes1-hydroxy-1-methyl-ethyl.

“Compounds of the present invention”, and equivalent expressions, aremeant to embrace compounds of Formula (I) as hereinbefore described.Reference to intermediates, whether or not they themselves are claimed,is meant to embrace their salts, N-oxides and solvates, where thecontext so permits.

“Halo” or “halogen” means fluoro, chloro, bromo, or iodo. Particularhalo or halogen is fluoro or chloro.

“Patient” includes human and other mammals.

“Pharmaceutically acceptable salts” refers to the non-toxic, inorganicand organic acid addition salts, and base addition salts, of compoundsof the present invention. These salts may be prepared in situ during thefinal isolation and purification of the compounds or by separatelyreacting the purified compound in its free base form with a suitableorganic or inorganic acid and isolating the salt thus formed. In somecases, the compounds themselves are capable of self-protonating basicsites on the molecule and forming an internal amphoteric salt.

“Suitable couple reagent” refers to a reagent suitable for reacting anamine with a carboxylic acid. Suitable coupling reagents include, butare not limited to, DMTMM, carbonyldiimidazole (CDI) and TBTU, DCC,phosphonium salts, and uronium salts.

Exemplary acid addition salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate,oleate, palmitate, stearate, laurate, borate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates,malonates, salicylates, propionates, methylene-bis-ß-hydroxynaphthoates,gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates and laurylsulfonate salts. See, for example S. M.Berge, et al., “Pharmaceutical Salts,” J. Pharm. Sci., 66, 1-19 (1977)that is incorporated herein by reference. Base addition salts can alsobe prepared by separately reacting the purified compound in its acidform with a suitable organic or inorganic base and isolating the saltthus formed. Base addition salts include pharmaceutically acceptablemetal and amine salts. Suitable metal salts include the sodium,potassium, calcium, barium, zinc, magnesium, and aluminum salts. Aparticular base addition salt is sodium salt or potassium salt. Suitableinorganic base addition salts are prepared from metal bases whichinclude sodium hydride, sodium hydroxide, potassium hydroxide, calciumhydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide,and zinc hydroxide. Suitable amine base addition salts are prepared fromamines which have sufficient basicity to form a stable salt, andparticularly include those amines which are frequently used in medicinalchemistry because of their low toxicity and acceptability for medicaluse. Ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine,ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine,diethanolamine, procaine, N-benzylphenethylamine, diethylamine,piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammoniumhydroxide, triethylamine, dibenzylamine, ephenamine,dehydroabietylamine, N-ethylpiperidine, benzylamine,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, ethylamine, basic amino acids, e.g., lysine andarginine, and dicyclohexylamine.

A particular embodiment of the invention is a compound of formula (I)wherein:

R1 is hydrogen;R2 is hydrogen; andR3 is hydroxyalkyl;or a pharmaceutically acceptable salt thereof.

Another particular embodiment of the invention is a compound of formula(I) wherein:

R1 is C₁-C₆alkyl;R2 is hydrogen; andR3 is hydroxyalkyl;or a pharmaceutically acceptable salt thereof.

Another particular embodiment of the invention is a compound of formula(I), which is:

-   2-Pyridin-2-yl-pyrimidine-5-carboxylic acid    3-5-(1-hydroxy-1methyl-ethyl)[1,2,4]oxadiazol-3-yl]benzyl amide;-   2-Pyridin-2-yl-pyrimidine-5-carboxylic acid    ((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amide;    or-   2-Pyridin-2-yl-pyrimidine-5-carboxylic acid    ((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amide;    or a pharmaceutically acceptable salt thereof.

It is to be understood that this invention covers all appropriatecombinations of the particular embodiments referred thereto.

The present invention also includes within its scope a pharmaceuticalcomposition comprising a pharmaceutically effective amount of a compoundof the invention, in admixture with a pharmaceutically acceptablecarrier.

Compounds of the present invention are PGDS inhibitors and thus, areuseful for treating allergic and/or inflammatory disorders, particularlydisorders such as allergic rhinitis, asthma, chronic obstructivepulmonary disease (COPD), chronic rhinosinusitus (CRS), and age-relatedmacular degeneration (AMD). Accordingly, another invention herein isdirected to a method of treating a patient suffering from allergicrhinitis, asthma, chronic obstructive pulmonary disease (COPD) and/orage-related macular degeneration (AMD) comprising administering to thepatient a pharmaceutically effective amount of compound of formula (I).

In addition to the indications and disorders recited above, PGDSinhibitors, including compounds of formula I, are useful for thetreatment of PGD2 mediated diseases including DP1, DP2, TP & PPAR gammaassociated diseases. Such diseases and disorders include the following:

1) Skin diseases including atopic dermatitis, chronic urticaria,flushing Proc Natl Acad Sci U.S.A. 2006 Apr. 25; 103(17):6682-7);2) Allergic diseases of the digestive system such as eosinophiliceosophagitis;3) Neurodegenerative diseases such as Alzheimer's and Krabbes disease(The Journal of Neuroscience, Apr. 19, 2006, 26(16):4383-4393);4) Muscle diseases such as Duchenne Muscular Dystrophy and Polymyositis(American Journal of Pathology. 2009; 174:1735-1744);5) Conditions associated with increased eosinophils or EosinophilicSyndrome;6) Diseases of the eye such as Uveitis, Graves Ophthalmopathy, allergicconjunctivitis and glaucoma;7) Vascular injury associated with diabetes such as diabetic retinopathyor with metabolic syndrome (Diabetes Res Clin Pract. 2007 June;76(3):358-67); and8) Bone diseases such as rheumatoid arthritis and ostcoarthritis (JRheumatol 2006; 33:1167-75).

References herein directed to treating should be understood to includeprophylactic therapy to inhibit PGDS, as well as to treat an establishedacute or chronic or physiological conditions associated with PGDS toessentially cure a patient suffering therefrom, or ameliorate thephysiological conditions associated therewith. Physiological conditionsdiscussed herein include some, but not all, of the possible clinicalsituations where an anti-allergic rhinitis and/or asthma treatment iswarranted. Those experienced in this field are well aware of thecircumstances requiring treatment.

In practice, the compound of the present invention may be administeredin pharmaceutically acceptable dosage form to humans and other mammalsby topical or systemic administration, including oral, inhalational,rectal, nasal, buccal, sublingual, vaginal, colonic, parenteral(including subcutaneous, intramuscular, intravenous, intradermal,intrathecal and epidural), intracisternal and intraperitoneal. It willbe appreciated that the particular route may vary with for example thephysiological condition of the recipient.

“Pharmaceutically acceptable dosage forms” refers to dosage forms of thecompound of the invention, and includes, for example, tablets, dragées,powders, elixirs, syrups, liquid preparations, including suspensions,sprays, inhalants tablets, lozenges, emulsions, solutions, granules,capsules and suppositories, as well as liquid preparations forinjections, including liposome preparations. Techniques and formulationsgenerally may be found in Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., latest edition.

A particular aspect of the invention provides for the compound of theinvention to be administered in the form of a pharmaceuticalcomposition.

Pharmaceutically acceptable carriers include at least one componentselected from the group comprising pharmaceutically acceptable carriers,diluents, coatings, adjuvants, excipients, or vehicles, such aspreserving agents, fillers, disintegrating agents, wetting agents,emulsifying agents, emulsion stabilizing agents, suspending agents,isotonic agents, sweetening agents, flavoring agents, perfuming agents,coloring agents, antibacterial agents, antifungal agents, othertherapeutic agents, lubricating agents, adsorption delaying or promotingagents, and dispensing agents, depending on the nature of the mode ofadministration and dosage forms.

Exemplary suspending agents include ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances.

Exemplary antibacterial and antifungal agents for the prevention of theaction of microorganisms include parabens, chlorobutanol, phenol, sorbicacid, and the like.

Exemplary isotonic agents include sugars, sodium chloride, and the like.

Exemplary adsorption delaying agents to prolong absorption includealuminum monostearate and gelatin.

Exemplary adsorption promoting agents to enhance absorption includedimethyl sulfoxide and related analogs.

Exemplary diluents, solvents, vehicles, solubilizing agents, emulsifiersand emulsion stabilizers, include water, chloroform, sucrose, ethanol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,tetrahydrofurfuryl alcohol, benzyl benzoate, polyols, propylene glycol,1,3-butylene glycol, glycerol, polyethylene glycols, dimethylformamide,Tween® 60, Span® 60, cetostearyl alcohol, myristyl alcohol, glycerylmono-stearate and sodium lauryl sulfate, fatty acid esters of sorbitan,vegetable oils (such as cottonseed oil, groundnut oil, olive oil, castoroil and sesame oil) and injectable organic esters such as ethyl oleate,and the like, or suitable mixtures of these substances.

Exemplary excipients include lactose, milk sugar, sodium citrate,calcium carbonate and dicalcium phosphate.

Exemplary disintegrating agents include starch, alginic acids andcertain complex silicates. Exemplary lubricants include magnesiumstearate, sodium lauryl sulfate, talc, as well as high molecular weightpolyethylene glycols.

The choice of pharmaceutical acceptable carrier is generally determinedin accordance with the chemical properties of the active compound suchas solubility, the particular mode of administration and the provisionsto be observed in pharmaceutical practice.

Pharmaceutical compositions of the present invention suitable for oraladministration may be presented as discrete units such as a solid dosageform, such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient, or as a powder orgranules; as a liquid dosage form such as a solution or a suspension inan aqueous liquid or a non-aqueous liquid, or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

“Solid dosage form” means the dosage form of the compound of theinvention is solid form, for example capsules, tablets, pills, powders,dragées or granules. In such solid dosage forms, the compound of theinvention is admixed with at least one inert customary excipient (orcarrier) such as sodium citrate or dicalcium phosphate or: (a) fillersor extenders, as for example, starches, lactose, sucrose, glucose,mannitol and silicic acid, (b) binders, as for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates andsodium carbonate, (e) solution retarders, as for example paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, (i)lubricants, as for example, talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, (j) opacifyingagents, (k) buffering agents, and agents which release the compound ofthe invention in a certain part of the intestinal tract in a delayedmanner.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Excipients such as lactose, sodium citrate, calciumcarbonate, dicalcium phosphate and disintegrating agents such as starch,alginic acids and certain complex silicates combined with lubricantssuch as magnesium stearate, sodium lauryl sulfate and talc may be used.A mixture of the powdered compounds moistened with an inert liquiddiluent may be molded in a suitable machine to make molded tablets. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the active ingredient therein.

Solid compositions may also be employed as fillers in soft andhard-filled gelatin capsules using such excipients as lactose or milksugar as well as high molecular weight polyethylene glycols, and thelike.

If desired, and for more effective distribution, the compound can bemicroencapsulated in, or attached to, a slow release or targeteddelivery systems such as a biocompatible, biodegradable polymer matrices(e.g., poly(d,l-lactide co-glycolide)), liposomes, and microspheres andsubcutaneously or intramuscularly injected by a technique calledsubcutaneous or intramuscular depot to provide continuous slow releaseof the compound(s) for a period of 2 weeks or longer. The compounds maybe sterilized, for example, by filtration through a bacteria-retainingfilter, or by incorporating sterilizing agents in the form of sterilesolid compositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use.

“Liquid dosage form” means the dose of the active compound to beadministered to the patient is in liquid form, for example,pharmaceutically acceptable emulsions, solutions, suspensions, syrupsand elixirs. In addition to the active compound, the liquid dosage formsmay contain inert diluents commonly used in the art, such solvents,solubilizing agents and emulsifiers.

When aqueous suspensions are used they can contain emulsifying agents oragents which facilitate suspension.

Pharmaceutical compositions suitable for topical administration meanformulations that are in a form suitable to be administered topically toa patient. The formulation may be presented as a topical ointment,salves, powders, sprays and inhalants, gels (water or alcohol based),creams, as is generally known in the art, or incorporated into a matrixbase for application in a patch, which would allow a controlled releaseof compound through the transdermal barrier. When formulated in anointment, the active ingredients may be employed with either aparaffinic or a water-miscible ointment base. Alternatively, the activeingredients may be formulated in a cream with an oil-in-water creambase. Formulations suitable for topical administration in the eyeinclude eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent for theactive ingredient. Formulations suitable for topical administration inthe mouth include lozenges comprising the active ingredient in aflavored basis, usually sucrose and acacia or tragacanth; pastillescomprising the active ingredient in an inert basis such as gelatin andglycerin, or sucrose and acacia; and mouthwashes comprising the activeingredient in a suitable liquid carrier.

The oily phase of the emulsion pharmaceutical composition may beconstituted from known ingredients, in a known manner. While the phasemay comprise merely an emulsifier (otherwise known as an emulgent), itdesirably comprises a mixture of at least one emulsifier with a fat oran oil or with both a fat and an oil. In a particular embodiment, ahydrophilic emulsifier is included together with a lipophilic emulsifierthat acts as a stabilizer. Together, the emulsifier(s) with, or without,stabilizer(s) make up the emulsifying wax, and together with the oil andfat make up the emulsifying ointment base which forms the oily dispersedphase of the cream formulations.

If desired, the aqueous phase of the cream base may include, forexample, a least 30% w/w of a polyhydric alcohol, i.e. an alcohol havingtwo or more hydroxyl groups such as, propylene glycol, butane 1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400)and mixtures thereof. The topical formulations may desirably include acompound that enhances absorption, or penetration of the activeingredient through the skin, or other affected areas.

The choice of suitable oils or fats for a composition is based onachieving the desired properties. Thus a cream should particularly be anon-greasy, non-staining and washable product with suitable consistencyto avoid leakage from tubes or other containers. Straight or branchedchain, mono- or dibasic alkyl esters such as di-isopropyl myristate,decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexylpalmitate or a blend of branched chain esters known as Crodamol CAP maybe used. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Pharmaceutical compositions suitable for rectal or vaginaladministrations mean formulations that are in a form suitable to beadministered rectally or vaginally to a patient and containing at leastone compound of the invention. Suppositories are a particular form forsuch formulations that can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax, which are solidat ordinary temperatures but liquid at body temperature and therefore,melt in the rectum or vaginal cavity and release the active component.

Pharmaceutical composition administered by injection may be bytransmuscular, intravenous, intraperitoneal, and/or subcutaneousinjection. The compositions of the present invention are formulated inliquid solutions, in particular in physiologically compatible bufferssuch as Hank's solution or Ringer's solution. In addition, thecompositions may be formulated in solid form and redissolved orsuspended immediately prior to use. Lyophilized forms are also included.The formulations are sterile and include emulsions, suspensions, aqueousand non-aqueous injection solutions, which may contain suspending agentsand thickening agents and anti-oxidants, buffers, bacteriostats andsolutes which render the formulation isotonic, and have a suitablyadjusted pH, with the blood of the intended recipient.

Pharmaceutical composition of the present invention suitable for nasalor inhalational administration means compositions that are in a formsuitable to be administered nasally or by inhalation to a patient. Thecomposition may contain a carrier, in a powder form, having a particlesize for example in the range 1 to 500 microns (including particle sizesin a range between 20 and 500 microns in increments of 5 microns such as30 microns, 35 microns, etc.). Suitable compositions wherein the carrieris a liquid, for administration as for example a nasal spray or as nasaldrops, include aqueous or oily solutions of the active ingredient.Compositions suitable for aerosol administration may be preparedaccording to conventional methods and may be delivered with othertherapeutic agents. Inhalational therapy is readily administered bymetered dose inhalers or any suitable dry powder inhaler, such as theEclipse, Spinhaler®, or Ultrahaler® as described in patent applicationWO2004/026380, and U.S. Pat. No. 5,176,132.

Actual dosage levels of active ingredient(s) in the compositions of theinvention may be varied so as to obtain an amount of activeingredient(s) that is (are) effective to obtain a desired therapeuticresponse for a particular composition and method of administration for apatient. A selected dosage level for any particular patient thereforedepends upon a variety of factors including the desired therapeuticeffect, on the route of administration, on the desired duration oftreatment, the etiology and severity of the disease, the patient'scondition, weight, sex, diet and age, the type and potency of eachactive ingredient, rates of absorption, metabolism and/or excretion andother factors.

Total daily dose of the compound of this invention administered to apatient in single or divided doses may be in amounts, for example, offrom about 0.001 to about 100 mg/kg body weight daily and particularly0.01 to 10 mg/kg/day. For example, in an adult, the doses are generallyfrom about 0.01 to about 100, particularly about 0.01 to about 10, mg/kgbody weight per day by inhalation, from about 0.01 to about 100,particularly 0.1 to 70, more especially 0.5 to 10, mg/kg body weight perday by oral administration, and from about 0.01 to about 50,particularly 0.01 to 10, mg/kg body weight per day by intravenousadministration.

The percentage of active ingredient in a composition may be varied,though it should constitute a proportion such that a suitable dosageshall be obtained. Dosage unit compositions may contain such amounts orsuch submultiples thereof as may be used to make up the daily dose.Obviously, several unit dosage forms may be administered at about thesame time. A dosage may be administered as frequently as necessary inorder to obtain the desired therapeutic effect. Some patients mayrespond rapidly to a higher or lower dose and may find much lowermaintenance doses adequate. For other patients, it may be necessary tohave long-term treatments at the rate of 1 to 4 doses per day, inaccordance with the physiological requirements of each particularpatient. It goes without saying that, for other patients, it will benecessary to prescribe not more than one or two doses per day.

The formulations can be prepared in unit dosage form by any of themethods well known in the art of pharmacy. Such methods include the stepof bringing into association the pharmaceutically active ingredient withthe carrier that constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials with elastomeric stoppers, and maybe stored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

Compounds of the invention may be prepared by the application oradaptation of known methods, by which is a meant method used heretoforeor described in the literature, for example those described by R. C.Larock in Comprehensive Organic Transformations, VCH publishers, 1989.

In the reactions described hereinafter it may be necessary to protectreactive functional groups, for example hydroxy, amino, imino, thio orcarboxy groups, where these are desired in the final product, to avoidtheir unwanted participation in the reactions. Conventional protectinggroups may be used in accordance with standard practice, for examplessee T. W. Greene and P. G. M. Wuts, Protecting Groups in OrganicSynthesis, 3rd edition, John Wiley & Sons, Inc., 1999.

A compound of formula (I) may be prepared (as shown in Scheme I below)by reacting a amine of type XI with a pyridylpyrimidinyl carboxylic acid(preparation shown in Scheme II) in the presence of a dehydratingcoupling reagent, such as DMTMM, in a variety of solvents including butnot limited to DMF. Suitable couple reagents include, but are notlimited to, DMTMM, carbonyldiimidazole (CDI) and TBTU, DCC, phosphoniumsalts, and uronium salts. A compound of formula (I) may also be prepared(as shown in Scheme Ia below) by direct coupling of an amine of type XIwith a pyridylpyrimidinyl ester (preparation shown in Scheme II) in thepresence of 0.1 to 1.0 equivalents of1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD). The reaction can be performedin the absence of solvent, or in the presence of added solvents,including, but not limited to ethers, esters, aromatic hydrocarbons. Theuse of strong bases other than TBD, including, but not limited to DBUand tetramethyl guanidine, also give product. The amine XI may beprepared through a process as detailed in Scheme III. A benzylic bromideVII may be reacted with Di-tert-butyl iminodicarboxylate in the presenceof bases including but not limited to cesium carbonate in a variety ofsolvents including but not limited to DMF to yield compounds VIII. Thesecompounds of type VIII may then be reacted with hydroxylamine (in thepresence of bases including but not limited to triethylamine in thecases where salts of hydroxylamine are used, such as hydroxylaminehydrochloride) in a variety of solvents including but not limited tomethanol to yield an amidoxime IX. The amidoxime may be reacted with acompound containing a carboxy functionality including but not limited toa methyl carboxylate in the presence of a base including but not limitedto potassium carbonate either in the presence or absence of a solventincluding but not limited to toluene (in certain cases the carboxyfunctionality may serve as a solvent for the reaction) to yield anoxadiazole X. The oxadiazole X may then be exposed to acidic conditionsincluding but not limited to hydrogen chloride in methanol to yield anamine XI. In the cases where R1 alkyl substitution is desired in amineXI, these amines may be prepared according to Scheme IV (either inenantioenriched or racemic form) using the tert-butyl sulfinamidemethodology developed by Ellman.

It will be appreciated that compounds of the present invention maycontain asymmetric centers. These asymmetric centers may independentlybe in either the R or S configuration. It will be apparent to thoseskilled in the art that certain compounds of the invention may alsoexhibit geometrical isomerism. It is to be understood that the presentinvention includes individual geometrical isomers and stereoisomers andmixtures thereof, including racemic mixtures, of compounds of Formula(I) hereinabove. Such isomers can be separated from their mixtures, bythe application or adaptation of known methods, for examplechromatographic techniques and recrystallization techniques, or they areseparately prepared from the appropriate isomers of their intermediates.

The compounds of the invention, their methods or preparation and theirbiological activity will appear more clearly from the examination of thefollowing examples that are presented as an illustration only and arenot to be considered as limiting the invention in its scope. Compoundsof the invention are identified, for example, by the followinganalytical methods.

Mass Spectra (MS) are recorded using a Micromass LCT mass spectrometer.The method is positive electrospray ionization, scanning mass m/z from100 to 1000.

300 MHz ¹H nuclear magnetic resonance spectra (¹H NMR) are recorded atambient temperature using a Varian Mercury (300 MHz) spectrometer withan ASW 5 mm probe. In the ¹H NMR chemical shifts (δ) are indicated inparts per million (ppm) with reference to tetramethylsilane (TMS) as theinternal standard.

As used in the examples and preparations that follow, as well as therest of the application, the terms used therein shall have the meaningsindicated: “kg”=kilograms, “g”=grams, “mg”=milligrams, “g”=micrograms,“mol”=moles, “mmol”=millimoles, “M”=molar, “mM”=millimolar,“M”=micromolar, “nM”=nanomolar, “L”=liters, “mL” or “ml”=milliliters,“L”=microliters, “° C.”=degrees Celsius, “mp” or “m.p.”=melting point,“bp” or “b.p.”=boiling point, “mm of Hg”=pressure in millimeters ofmercury, “cm”=centimeters, “nm”=nanometers, “abs.”=absolute,“conc.”=concentrated, “c”=concentration in g/mL, “rt”=room temperature,“TLC”=thin layer chromatography, “HPLC”=high performance liquidchromatography, “i.p.”=intraperitoneally, “i.v.”=intravenously,“s”=singlet, “d”=doublet; “t”=triplet; “q”=quartet; “m”=multiplet,“dd”=doublet of doublets; “br”=broad, “LC”=liquid chromatograph,“MS”=mass spectrograph, “ESI/MS”=electrospray ionization/massspectrograph, “R_(T)”=retention time, “M”=molecular ion, “PSI”=poundsper square inch, “DMSO”=dimethyl sulfoxide, “DMF”=N,N-dimethylformamide,“DCM”=dichloromethane, “HCl”=hydrochloric acid, “SPA”=ScintillationProximity Assay, “EtOAc”=ethyl acetate, “PBS”=Phosphate Buffered Saline,“IUPAC”=International Union of Pure and Applied Chemistry,“MHz”=megahertz, “MeOH”=methanol, “N”=normality. “THF”=tetrahydrofuran.“min”=minute(s), “N₂”=nitrogen gas, “MeCN” or “CH₃CN”=acetonitrile,“Et₂O”=ethyl ether, “TFA”=trifluoroacetic acid, “˜”=approximately,“MgSO₄”=magnesium sulfate, “Na₂SO₄”=sodium sulfate, “NaHCO₃”=sodiumbicarbonate, “Na₂CO₃”=sodium carbonate, “MCPBA”=3-Chloroperoxybenzoicacid, “NMP”=N-methylpyrrolidone, “PS-DCC”=polymersupported-dicyclohexylcarbodiimide, “LiOH”=Lithium hydroxide,“PS-trisamine”=polymer supported-trisamine, “PGH2”=prostaglandin H2,“PGD2”=prostaglandin D2; “PGE2”=prostaglandin E2, “hPGDS”=HematopoieticPGD2 Synthase, “GSH”=glutathione (reduced), “EIA”=Enzyme immunoassay,“KH₂PO₄”=potassium phosphate, monobasic, “K₂HPO₄”=potassium phosphate,dibasic, “FeCl₂”=ferrous chloride, “MOX”=methoxylamine; “EtOH”=ethanol,“DMSO”=dimethylsulfoxide, “Ag₂O”=silver(I) oxide,“HATU”=O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, “HOAt”=1-hydroxy-7-azabenzotriazole,“DIPEA”=N,N-diisopropylethylamine,“HOTT”=S-(1-Oxido-2-pyridyl)-N,N,N,N′-tetramethylthiuroniumhexafluorophosphate,“HCTU”=N,N,N′,N′-tetramethyl-O-(6-chloro-1H-benzotriazol-1-yl)uroniumhexafluorophosphate, “PyBrOP”=bromo-tris-pyrrolidinophosphoniumhexafluorophosphate, “LiAlH4”=lithium aluminum hydride,“PyAOP”=(7-azabenzotriazol-1-yloxy)-tripyrrolidinophosphoniumhexafluorophosphate,“TBTU”=O-benzotriazol-1-yl-N,N,N,N,-tetramethyluroniumtetrafluoroborate, “NaHMDS”=sodium bis(trimethylsilyl)amide,“NMP”=N-methyl-2-pyrrolidinone, “HOSA”=hydroxylamine-O-sulfonic acid,“DMTMM”=4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumchloride, “TMSN₃”=trimethylsilyl azide, “TBAF”=tetrabutylammoniumfluoride, “TFAA”=trifluoro acetic anhydride.

EXAMPLES

Following procedures similar to those described in the above examples,the following compounds are made:

Example 1 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-benzylamide

Step 1

3-Bromomethyl-benzonitrile (42.9 g, 219 mmol, 1 equivalent) is combinedwith Di-tert-butyl iminodicarboxylate (50 g, 230.13 mmol, 1.05equivalents) and cesium carbonate (74.98 g, 230.13 mmol, 1.05equivalents) in N,N-dimethyl formamide (DMF) (230 mL). The reaction isstirred at room temperature overnight and then partitioned betweendiethyl ether (500 mL) and water (1 L). The aqueous layer is extractedwith an additional portion of diethyl ether (250 mL) and the combinedether layers are washed with brine (2×200 mL). The organic layer is thendried (MgSO₄), filtered, and reduced in vacuo to yield oil that slowlycrystallized to give 2-[(3-cyanophenyl)methyl]-imidodicarbonic acid1,3-bis(1,1-dimethylethyl) ester (72 g, 99%). MS: 333 (M+H), 355 (M+Na).

¹H NMR (300 MHz, CDCl₃): δ=1.47 (s, 18H), 4.79 (s, 2H), 7.42 (t, 1H),7.54-7.60 (m, 3H).

Step 2

Hydroxylamine hydrochloride (23.43 g, 375 mmol, 2.5 equivalents) isadded to a solution of 2-[(3-cyanophenyl)methyl]-imidodicarbonic acid1,3-bis(1,1-dimethylethyl) ester (50 g, 150 mmol, 1 equivalent) inmethanol (450 mL) and the mixture is chilled in an ice water bath.Triethylamine (37.87 g, 375 mmol, 2.5 equivalents) is added and thereaction mixture is allowed to stir overnight, warming up to roomtemperature slowly as the bath thaws. The reaction is then reduced invacuo and the residue partitioned between ethyl acetate (1 L) and water(500 mL). The water layer is extracted with an additional portion ofethyl acetate (200 mL) and the combined organic layers are washed withbrine (200 mL), dried over sodium sulfate, and filtered. At this point,heptane and toluene (100 mL each) are added and the reaction reduced invacuo to yield 2-[[3-[(hydroxyamino)iminomethyl]phenyl]methyl]-imidodicarbonic acid1,3-bis(1,1-dimethylethyl) ester as a clear gel (54.7 g (>99%), that isused directly without further purification.

Step 3

Potassium carbonate (4.35 g, 31.46 mmol, 1.15 equivalents) is added to aflask charged with 2-[[3-[(hydroxyamino)iminomethyl]phenyl]methyl]-imidodicarbonic acid1,3-bis(1,1-dimethylethyl) ester (10 g, 27.36 mmol, 1 equivalent) fromstep 2 in Toluene (30 mL), followed by 2-Hydroxy-2-methyl-propionic acidmethyl ester (3.716 g, 31.46 mmol, 1.15 equivalents). The reaction isheated to reflux. After 48 hours the reaction is partitioned betweenEtOAc (300 mL) and water (200 mL). The EtOAc is washed with Brine (100mL), dried over sodium sulfate, filtered and then reduced in vacuo toyield a residue that is taken on directly.

A solution of 4N HCl in dioxane (60 mL) is added to an ice chilledmixture of the residue (27 mmol) from the previous reaction in p-dioxane(60 mL). The ice water bath is removed and the reaction is allowed towarm to room temperature. After 6 hours, the reaction is diluted withdiethyl ether (200 mL). The white solid is collected via filtration,washed with diethyl ether (˜50 mL) and then dried in vacuo to yield3-[5-(1-Hydroxy-1-methyl-ethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-aminehydrochloride (5.84 g, 79% over two steps). MS: 234 (M+H). ¹H NMR (300MHz, DMSO): δ=1.626 (s, 6H), 4.13-4.15 (d, 2H), 6.11 (bs, 1H), 7.62 (t,1H), 7.72 (d, 1H), 8.02 (d, 1H), 8.15 (s, 1H), 8.45 (bs, 3H).

Step 4

N-methyl morpholine (NMM) (1.12 g, 11.12 mmol, 1 equivalent) is added toa mixture of 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid (2.24 g, 11.12mmol, 1 equivalent) and3-[5-(1-Hydroxy-1-methyl-ethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-aminehydrochloride (3 g, 11.12 mmol, 1 equivalent) in DMF (50 mL). Afterstirring at room temperature for 5 minutes,4-(4,6-Dimethoxy-[1,3,5]triazin-2-yl)-4-methyl-morpholin-4-ium chloride(DMTMM) (3.08 g, 11.12 mmol, 1 equivalent) is added and the reactionstirred at room temperature, for 3 hours.

The reaction is diluted into ice water (500 mL) and the suspension isextracted with EtOAc (2×300 mL). The combined ethyl acetate layers arewashed with brine (2×100 mL), dried over sodium sulfate, and reduced invacuo to give crude product which is recrystallized using ethylacetate/ethanol to yield 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-benzylamide aswhite crystalline solid (1.95 g, 42%) Note: Yields are variabledepending on the purity of the coupling partners and solvents used forrecrystallization. MS: 417 (M+H). ¹H NMR (300 MHz, DMSO): δ=1.62 (s,6H), 4.65 (d, 2H), 6.08 (s, 1H), 7.54-7.63 (m, 3H), 7.93 (d, 1H),7.99-8.04 (m, 2H), 8.45 (d, 1H), 8.79 (d, 1H), 9.37 (s, 2H), 9.57 (t,1H).

Alternatively, the coupling may be achieved using CDI(carbonyldiimidazole) or TBTU. The coupling shown below may be done in,for example, DMF and/or THF.

To a 5 L jacketed reactor was added 68.89 g of the carboxylic acid andabout 346 ml DMF. To this slurry was added 74.9 g of the CDI at 22±2° C.The amine (79.87 g) was dissolved in about 69 mL DMF and added over 8minutes. This turned the thick slurry into a clear yellow/brownsolution. The temperature increased to 35° C. Heptane (202 ml) was addedfollowed by water (596 ml) slowly over 20 minutes. During wateraddition, the temperature increased from 22 to 33° C. As the reactionmixture was stirred, crystals began to form. Water (5.15 L) was added.The reaction mixture was filtered on a 185 mm diameter Buchner andwashed with 2×750 mL water. The cake was collected and dried undervacuum (45° C., 100 mbar pressure, nitrogen flush) to yield 122.15 g ofproduct.

HPLC method: Eclipse XDB phenyl column, 3.5 micron, 4.6×150 mm,detection at 254 nm, gradient: started at 5:95:0.1% ACN/water/TFA thenramped over 8 min to 70:30:0.1% ACN/water/TFA, held 4.5 min; productretention time: 6.5 min.

Alternatively, the coupling may proceed via the acid chloride as shownbelow.

A 100 mL 3 neck round bottom flask equipped with magnetic stirring,temperature controller, and Firestone Valve (N2) was charged with2-[3-(3-aminomethylphenyl)-[1,2,4]-oxadiazol-5-yl]-propan-2-ol free base(600 mg, 2.57 mmole, 1 eq), NMP (5 mL) and triethylamine (2.25 mL).2-Pyridin-2-yl-pyrimidine-5-carbonyl chloride HCl (0.7 g, 2.7 mmole, ca96% acid) was added. The reaction was quenched after about 2.5 hours byadding toluene (5 mL) and water (5×10 mL). The reaction was filtered andthe cake was washed with toluene and water to yield a solid (0.85 g, 79%yield).

¹H NMR (300 MHz, d₆-DMSO): δ=1.61 (s, 6H), 4.64 (d, 2H), 6.08 (s, 1H),7.6 (m, 3H), 7.95 (d, 1H), 8.04 (m, 2H), 8.45 (d, 1H), 8.8 (d, 1H), 9.37(s, 1H), 9.57 (t, 1H)

Example 1a Alternative Synthesis for3-[5-(1-Hydroxy-1-methyl-ethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-aminehydrochloride

Scheme V—Step 1

A 5-L jacketed glass reactor equipped with an overhead mechanicalstirrer, a thermocouple probe and a nitrogen purge was charged at 20-25°C. with 3-cyanobenzaldehyde (100.0 g, 0.763 mol, 1.0 eq.) and ethanol(200 proof) (394.5 g, 500 mL, 5 v/w parts). To the suspension wascharged via addition funnel, a solution of hydroxylamine hydrochloride(159.0 g, 2.288 mole, 3.0 eq.) in water (250 mL, 2.5 parts) over aperiod of 30-45 min while maintaining temperature of 20-25° C. Theaddition funnel was rinsed with water (20 mL) and the rinse was added tothe reactor. After addition of ca 45 mL of NH₂OH.HCl solution, the soliddissolved to provide a clear solution. Within 10 min the solution turnedcloudy and a solid crystallized to provide a suspension. The solid isbelieved to be the oxime resulting from addition of hydroxylamine to thealdehyde function. The suspension was stirred at 20-25° C. for 1 h. Tothe suspension was charged via a addition funnel, a solution of sodiumcarbonate (121.25 g, 1.144 mole, 1.5 eq,) in water (390 mL, 3.9 parts)over a period of 1.5-2.0 h while maintaining a temperature of 20-22° C.The addition funnel was rinsed with water (20 mL) and the rinse wasadded to the reactor. Evolution of CO₂ was observed. The suspension washeated to 29-30° C. and stirred at 29-30° C. for 24 h. Water (1.32 L,13.2 parts) was charged to the reactor over 45-60 min while maintaininga temperature of 30-32° C. The suspension was heated to and held at76-78° C. for 30-60 min to get a clear solution. The solution was cooledto 55-60° C. over 90 min. Product crystallized at 55-60° C. Thesuspension was stirred at 55-60° C. for 60 min. The suspension wascooled to 20-22° C. over 8-12 h. The suspension was cooled to 2-5° C.and stirred at 2-5° C. for 4 h. The suspension was filtered (Buchnerfunnel, 14.5 cm o.d.) and the cake was washed with water (250 mL, 2.5parts). The cake was dried under suction for 5 h. The cake wastransferred to a drying dish and dried under vacuum (25-50 torr, 50° C.,N₂) for 60 h to provide 127.33 g (93.2% yield) of product as a whitecrystalline solid with a purity of 99.9% (HPLC).

HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron, 4.6×150 mm, 25° C.,detection at 240 nm, gradient: 5:95:0.1 CH₃CN/H₂O/TFA isocratic 2 minthen ramped over 16 min to 90:10:0.1 CH₃CN/H₂O/TFA; product retentiontime: 3.6-4.4 min (three peaks)

Scheme V—Step 2

A 5-L jacketed glass reactor equipped with an overhead mechanicalstirrer, a thermocouple probe and a nitrogen purge was charged at 22-27°C. with (N-hydroxy-3-hydroxyiminomethyl)benzamidine) (100.0 g, 0.558mol, 1.0 eq.) and 1-methyl-2-pyrrolidinone (NMP) (267.3 g, 260 mL, 2.6v/w parts). To the suspension was charged via addition funnel methyl2-hydroxyisobutyrate (197.8 g, 1.674 mole, 3.0 eq.) over 15-30 min whilemaintaining temperature of 25-27° C. The mixture was stirred at 25-27°C. for 30-45 min to get a clear solution. To the solution was chargedvia an addition funnel 25 w % sodium methoxide solution in methanol(361.7 g, 1.674 mole, 3.0 eq.) over 30-60 min while maintaining atemperature of 25-27° C. The solution was heated at 29-30° C. for 7hours. After 30-45 min at 29-30° C., the solution turned into asuspension. Water (1.8 L, 18 parts) was charged via an addition funnelover 30-60 min while maintaining a temperature of 22-25° C. Thesuspension dissolved to provide a clear solution with a pH 12.2 (pHmeter). The pH of the solution was adjusted to 5.0 by charginghydrochloric acid (37.1 w %) (77.4 g, 0.787 mole, 1.4 eq) over 30-45 minwhile maintaining a temperature of 22-25° C. The product crystallizedupon acidification with hydrochloric acid. After cooling to 5-10° C. andstirring at 5-10° C. for 2 h, the suspension was filtered (Buchnerfunnel, 27.5 cm i.d.), and the cake was washed with water (700 mL, 7parts) and dried under suction for 7 h. The cake was transferred to adrying dish and dried under vacuum (25-50 torr, 50° C., N2) for 20-24 hto provide 132.0 g (95.6% yield) of product as a white crystalline solidwith a purity of 99.7% (HPLC).

HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron, 4.6×150 mm, 25° C.,detection at 240 nm, gradient: 5:95:0.1 CH₃CN/H₂O/TFA isocratic 2 minthen ramped over 16 min to 90:10:0.1 CH₃CN/H₂O/TFA; product retentiontime: 10.8 min

Scheme V—Step 3

A 5-L jacketed glass reactor equipped with an overhead mechanicalstirrer, a thermocouple probe and a nitrogen purge was charged at 20-25°C. with3-[5-(1-hydroxy-1-methylethyl)-[1,2,4]oxadiazol-3-yl]benzaldehyde oxime(100.0 g, 0.404 mol, 1.0 eq.) and glacial acetic acid (1888.2 g, 1.8 L,18 v/w parts). The suspension was heated to 28-30° C. and stirred till aclear solution was obtained (30-45 min). The solution was cooled to22-24° C. and zinc dust (105.8 g, 1.618 mole, 4.0 eq.) was added via anaddition funnel over 90-120 min while maintaining a temperature of22-26° C. Note: Zinc dust addition was exothermic. The suspension wasstirred at 24-26° C. for 2-3 hours. The suspension was filtered under N₂(an inverted funnel with N₂ supply) through Celite (40 g). The solidswere washed with EtOH (200 proof)/H₂O (1/1, 894.5 g, 1 L, 10 parts) andEtOH (200 proof) (250 mL, 197.3 g, 2.5 parts). The filtrate wastransferred to a 5-L reactor and concentrated under reduced pressure(45-50 torr, 44-47° C., jacket temperature 50-55° C.) to a volume of ca.350 mL (3.5 parts). The vacuum was broken with N₂ and the reactor wascooled to 22° C. The mixture was a thick suspension. Toluene (2162.5 g,2.5 L, 25 parts) was charged to the reactor and suspension wasconcentrated under reduced pressure (70-75 torr, 42-47° C., jackettemperature 50-55° C.) to a volume of ca 350 mL (3.5 parts). Vacuum wasbroken with N₂ and the reactor was charged with toluene (129.8 g, 150mL, 1.5 parts) at 22° C. The suspension was stirred at 22° C. for 15-20minutes and the phases were allowed to separate. The upper layer ismainly toluene and the lower layer contains the acetate salt of desiredproduct.

HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron, 4.6×150 mm, 25° C.,detection at 240 nm, gradient: 5:95:0.1 CH₃CN/H₂O/TFA isocratic 2 minthen ramped over 16 min to 90:10:0.1 CH₃CN/H₂O/TFA; product retentiontime: 7.9 min

Scheme V—Step 4a

2-MeTHF (1290.0 g, 1.5 L, 15 parts) was added to the reactor. Aqueousammonium hydroxide (29.5 w %) (353.8 g, 400 mL, 4 parts) was charged viaan addition funnel over 30-45 min while maintaining a temperature of20-25° C. The mixture was stirred at 22-25° C. for 30-45 min and thephases were allowed to separate. The pH of the aqueous phase should bebasic (pH observed 10.9). The organic phase was washed with 15.3 w %aqueous sodium chloride (2×442.1 g, 2×400 mL, 2×4 parts). Note: 15.3 w %aq. NaCl was prepared by dissolving NaCl (180 g) in water (1000 g). Theorganic phase was concentrated under reduced pressure (100-110 torr,30-34° C., jacket temperature 35-40° C.) to a volume of ca 900 mL (9parts). Vacuum was broken with N₂ and the solution was filtered toremove a small amount of NaCl (ca 400 mg). The funnel was rinsed with2-MeTHF (86.0 g, 100 mL, 1 part) to provide a solution of2-[3-(3-aminomethylphenyl)-[1,2,4]-oxadiazol-5-yl]-propan-2-ol free basein 2-MeTHF/toluene (899.0 g, 1 L, 10 parts). Assay (w/w) of the solutionprovided the product (83.61 g, 9.3 w %) in 88.7% yield with a purity of95.1 A % (HPLC); 2-MeTHF 68.7 w % and toluene 21.2 w %.

HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron, 4.6×150 mm, 25° C.,detection at 240 nm, gradient: 5:95:0.1 CH₃CN/H₂O/TFA isocratic 2 minthen ramped over 16 min to 90:10:0.1 CH₃CN/H₂O/TFA; product retentiontime: 7.8 min

Scheme V—Step 4

A 5 L reactor equipped with mechanical stirrer, thermocouple probe andN2 inlet was charged with THF (1.5 L) and2-[3-(3-aminomethylphenyl)-[1,2,4]-oxadiazol-5-yl]-propan-2-ol AcOH(111.27 g). The solution turned into a suspension. A solution of Na₂CO₃(85.73 g) in water (600 ml) was added slowly with cooling (thermocoupleat 15° C.). The reaction was stirred at room temperature for about 10minutes. Di-tert-butyl dicarbonate (97.1 g) in THF (90 mL) was added viaa dropping funnel over about 12 minutes with cooling (thermocouple setto 15° C.). The reaction mixture was warmed (thermocouple set to 22°C.). The mixture separates into two distinct layers after firstappearing as a suspension and then becomes a suspension again. Ethylacetate (750 mL) was added and the suspension was stirred for 15 minutesat room temperature. Celite (545 (25 g) was added to the reactor and themixture was stirred for 15 minutes. The slurry was transferred to a 4 LErlenmeyer flask. It was filtered through Celite 545 (sintered glassfunnel, Kimax 2000 mL-125 C charged with 100 g of Celite 545). TheCelite/zinc salts were washed with ethyl acetate (500 mL). The organiclayer was collected and washed with 1/1 H2O/sat. aq NaCl (2×500 mL), pHof aqueous layer 5-7. The filtrate was charged to a clean reactor andthe reactor was fitted with a one-piece distillation apparatus (P=250torr, Δp=5 torr, thermocouple set to 40° C.). When the volume of liquidin reactor was about 250 mL, the pressure was equalized with N2 and thereaction was cooled (thermocouple set to 22° C.). The reactor wascharged with ethyl acetate (1500 mL). Resumed distillation (P=180-200torr, Δp=5 torr, thermocouple set to 50° C.) till volume of solution inthe reactor was ca 500 mL. The pressure was equalized with N2 and thereaction was cooled (thermocouple set to 22° C.). Yield of{3-[5-(1-hydroxy-1-methylethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-carbamicacid tert-butyl ester=126.46 g (quant., solution in ethyl acetate). Thesolution was used in step 5

HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron, 4.6×150 mm, 25° C.,detection at 240 nm, gradient: 5:95:0.1 CH₃CN/H₂O/TFA isocratic 2 minthen ramped over 16 min to 90:10:0.1 CH₃CN/H₂O/TFA; product retentiontime: 13.8 min

Scheme V—Step 5

A 5 L reactor equipped with a mechanical stirrer, thermocouple and N2inlet was charged with{3-[5-(1-hydroxy-1-methylethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-carbamicacid tert-butyl ester (126.46 g) as solution in ethyl acetate (from step4). The solution was cooled (3-15° C.). Added HCl gas (102 g) from alecture bottle over 30 minutes. The reaction was warmed to 15° C. over45 minutes and a slurry was formed. This slurry was transferred to anErlenmeyer flask (1 L). The contents were then filtered using a Buchnerfunnel. The cake was rinsed with ethyl acetate (350 mL) and suctiondried. The solid was then transferred to a drying dish and dried (0.9″Hg, 35 C, N2) to yield 83.52 g of a solid (76.6% overall yield steps3-5).

HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron, 4.6×150 mm, 25° C.,detection at 240 nm, gradient: 5:95:0.1 CH₃CN/H₂O/TFA isocratic 2 minthen ramped over 16 min to 90:10:0.1 CH₃CN/H₂O/TFA; product retentiontime: 8.0 min

Example 1b

A reactor with stirring and a nitrogen blanket was charged with 2-Me-THF(5 mL), the ester (500 mg), the benzylamine (545 mg) and1,5,7-triazabicyclo[4,4,0]dec-5-ene (TBD) (97.5 mg, 0.3 eq) to yield ayellowish suspension. The reactor was positioned into a heating blockthat was preheated to 79° C. The reaction was stirred for about 3 hours,then was removed from the block, allowed to cool to room temperature,then placed in an ice bath, stirred 15 minutes, and filtered. Thereactor and cake were rinsed with 1 mL cold 2-Me-THF. The white cakedwas rinsed with 5×2 mL water at room temperature and suction dried for1.5 hours. The white solid (0.77 g) was transferred to an oven andheated at 70° C. (N2, 45 mbar) overnight. Yield: 750 mg, 77%.

Alternative work up: After a reaction employing 2-Me-THF (4 mL), theester (300 mg), the benzylamine (327 mg) and1,5,7-triazabicyclo[4,4,0]dec-5-ene (TBD) (58.5 mg, 0.3 eq) wascomplete, the mixture was partitioned with 2 mL of water and cooled. Theorganic phase was separated, diluted with 2 mL of 2-Me-THF, then waswashed with 5 mL of water. The combine aqueous phase was extracted with2 mL of 2-Me-THF. The combined organic phase was concentrated and dried.Yield: 0.57 g, 97%.

HPLC method: Eclipse XDB C8 column, 5 micron, 4.6×150 mm, 35° C.,detection at 270 nm, gradient: 5:95:0.1% ACN/water/TFA held 5 min thenramped over 7 min to 50:50:0.1% ACN/water/TFA, held 3 min: productretention time: 12.9 min.

Example 2 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amide

Step 1

3-Acetylbenzonitrile (5 g, 34.4 mmol) is added to a flask containing(R)-(+)-2-Methyl-2-propanesulfinamide (3.48 g, 28.7 mmol) and titanium(IV) ethoxide (13.1 g, 57.4 mmol) in THF (70 mL) and the reactionmixture heated at 75° C. overnight. The reaction mixture is cooled (−48°C.) and L-Selectride (1M solution in THF, 57.4 mL) added dropwise over 1hour. The reaction stirred for 2 hrs and allowed to warm to roomtemperature. The reaction is then cooled to 0° C. and methanol (3 mL)added. Brine (150 mL) is added with stirring and the suspension filteredthrough Celite. The crude material is extracted with ethyl acetate,dried (MgSO₄), filtered and evaporated under vacuum. The cruse ispurified by column chromatography eluting with heptane-ethyl acetate togiveN-[(1S)-1-(3-cyanophenyl)ethyl]-2-methyl-[S(R)]-2-propanesulfinamide(78%)

MS: 251 (M+H)

¹H NMR (300 MHz, CDCl₃): δ=1.22 (s, 9H), 1.54 (d, 3H), 3.36 (bs, 1H),4.55-4.7 (m, 1H), 7.43 (d, 1H), 7.46 (d, 1H), 7.56-7.6 (m, 2H), 7.64 (s,1H).

Step 2

N-Hydroxy-3-[(S)-1-(2-methyl-propane-2-sulfinylamino)-ethyl]-benzamidine

Hydroxylamine hydrochloride (3.43 g, 55 mmol) and methanol (70 mL) areadded to a flask containingN-[(1S)-1-(3-cyanophenyl)ethyl]-2-methyl-[S(R)]-2-propanesulfinamide(5.5 g, 22 mmol) and the suspension cooled in an ice water bath.Triethyl amine (5.55 g, 55 mmol) is added to the flask and the reactionmixture is allowed to warm to room temperature over night. The reactionmixture evaporated under reduced pressure and the crude partitionedbetween water and DCM. Organic layer separated, dried (Na₂SO₄) andevaporated under reduced pressure to giveN-Hydroxy-3-[(S)-1-(2-methyl-propane-2-sulfinylamino)-ethyl]-benzamidine(5.48 g).

MS: 284 (M+H). ¹H NMR (300 MHz, CDCl₃): δ=1.21 (s, 9H), 1.52 (s, 3H),3.33 (s, 1H), 3.77 (bs, 1H), 4.59-4.61 (m, 1H), 4.88 (1H, bs), 7.35-7.37(m, 2H), 7.50-7.52 (m, 1H), 7.64 (s, 1H)

Step 3

2-Methyl-propane-2-sulfinic acid((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amide

Methyl 2-Hydroxy-2-methyl-propionate (20 mL) and K₂CO₃ (806 mg, 5.8mmol) are added to a flask containingN-Hydroxy-3-[(S)-1-(2-methyl-propane-2-sulfinylamino)-ethyl]-benzamidine(1.5 g, 5.3 mmol) and heated under reflux for 6 hrs. The reactionmixture is evaporated under reduced pressure and partitioned betweenwater and ethyl acetate. The organic layer separated, dried (Na₂SO₄) andpurified by flash column chromatography eluting with heptane-ethylacetate mixture to give 2-Methyl-propane-2-sulfinic acid((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amide(1.05 g).

MS: 352 (M+H).

¹H NMR (300 MHz, CDCl₃): δ=1.22 (s, 9H), 1.58 (d, 3H), 1.75 (s, 6H),3.48 (bs, 1H), 4.65 (m, 1H), 7.45-7.47 (m, 2H), 8.01 (m, 1H), 8.08 (s,1H)

Step 4

2-{3-[3-((S)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl}-propan-2-olHydrochloride

Hydrogen chloride in p-dioxane (4N, 1.42 mL) is added to a cooledsolution of 2-Methyl-propane-2-sulfinic acid((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl-phenyl}-ethyl)-amide(1 g, 2.85 mmol) in methanol (3 mL) at 0° C. and stirred for 20 min.Diethyl ether (30 mL) is added, decanted, and the residue is washed withanother aliquot of diethyl ether. The residue is dried in vacuo to yield2-f 3-[3-((S)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl]-propan-2-olHydrochloride (560 mg).

MS: 231 (ES+, —OH ionized)

¹H NMR (300 MHz, DMSO): δ=1.55 (d, 3H), 1.63 (s, 6H), 4.53-4.57 (m, 1H),6.1 (bs, 1H), 7.64 (t, 1H), 7.76 (d, 1H), 8.01 (d, 1H), 8.15 (s, 1H),8.56 (bs, 2H)

Step 5

N-methyl morpholine (NMM) (196 mg, 1.94 mmol) is added to a mixture of2-Pyridin-2-yl-pyrimidine-5-carboxylic acid (390 mg, 1.94 mmol) and2-[3-[3-((S)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl]-propan-2-olHydrochloride (550 mg, 1.94 mmol) in DMF (20 mL). After stirring at roomtemperature for 5 minutes,4-(4,6-Dimethoxy-[1,3,5]triazin-2-yl)-4-methyl-morpholin-4-ium chloride(DMTMM) (537 mg, 1.94 mmol) is added and the reaction stirred at roomtemperature for 2 hours. The reaction mixture poured on to ice water andthe suspension is extracted with EtOAc (7×100 mL). The combined ethylacetate layer is washed with brine (50 mL), dried over sodium sulfate,and reduced in vacuo to give crude product which is purified by HPLC(C18 column) eluting with acetonitrile-water mixture to give2-Pyridin-2-yl-pyrimidine-5-carboxylic acid((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amideas amorphous glass (650 mg, 78%).

MS: 431 (M+H).

¹H NMR (300 MHz, DMSO): δ=1.58 (d, 3H), 1.62 (s, 6H), 5.3 (m, 1H), 7.56(t, 1H), 7.7 (d, 1H), 7.92 (m, 2H), 8.08 (s, 1H), 8.43 (t, 1H), 8.72 (d,1H), 8.9 (d, 1H), 9.47 (s, 2H), 9.59 (d, 1H).

[α]_(d) (Methanol)=+57.2°

Example 3 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amide

Step 1

Potassium hydrogen sulfate (13.6 g, 100 mmol) is added to a mixture of3-Formylbenzonitrile (7.21 g, 55 mmol) and(S)-(+)-2-Methyl-2-propanesulfinamide (6.06 g, 50 mmol) in toluene (500mL) and heated at 45° C. for 2 days. The reaction mixture is filtered,the filtrate evaporated under reduced pressure and purified by columnchromatography eluting with ethyl acetate-heptane mixture to give,N-[(3-cyanophenyl)methylene]-2-methyl-, [S(S)]-2-Propanesulfinamide(9.65 g)

MS: 235 (M+H).

¹H NMR (300 MHz, CDCl₃): δ=1.29 (s, 91H), 7.62 (t, 1H), 7.79 (d, 1H),8.04 (d, 1H), 8.17 (bs, 1H), 8.60 (s, 1H).

Step 2

Methyl magnesium bromide (34.3 mL of 3M solution in diethyl ether, 102.9mmol) is added over 30 minutes to a solution ofN-[(3-cyanophenyl)methylene]-2-methyl-, [S(S)]-2-Propanesulfinamide(9.65 g, 41.18 mmol) in DCM (200 mL) at −45° C. and stirred at thattemperature for 4 hrs. The cooling bath is then removed, allowed to warmup to −10° C. and quenched with saturated NaHCO₃ (250 mL). The organiclayer is separated and the aqueous layer is extracted with more DCM (100mL). The organic extracts are combined, dried (Na₂SO₄) and evaporatedunder reduced pressure to give 2-Methyl-propane-2-sulfinic acid[(R)-1-(3-cyano-phenyl)-ethyl]-amide as the major product.

MS: 251 (M+H).

¹H NMR (300 MHz, CDCl₃): δ=1.22 (s, 9H), 1.54 (d, 3H), 3.35 (s, 1H),4.56-4.65 (m, 1H), 7.42-7.48 (m, 1H), 7.56-7.59 (m, 2H), 7.64 (s, 1H).

Step 3

Hydrogen chloride (4N in p-dioxane, 21 mL) is added to a solution of2-Methyl-propane-2-sulfinic acid [(R)-1-(3-cyano-phenyl)-ethyl]-amide(10.29 g, 41.1 mmol) in methanol (21 mL) and stirred at room temperaturefor 40 minutes. Reaction mixture is then evaporated under reducedpressure and the crude triturated with diethyl ether to give an offwhite solid which is crystallized from Methyl t-butyl ether and ethanolmixture to give 3-((R)-1-Amino-ethyl)-benzonitrile hydrochloride as themajor product.

MS: 147 (M+H).

¹H NMR (300 MHz, DMSO): δ=1.53 (d, 3H), 4.45-4.52 (m, 1H), 7.65 (t, 1H),7.84-7.91 (m, 2H), 8.03 (s, 1H), 8.67 (bs, 3H).

Step 4

N-methyl morpholine (NMM) (1.01 g, 10 mmol) is added to a mixture of2-Pyridin-2-yl-pyrimidine-5-carboxylic acid (2 g, 10 mmol) and3-((R)-1-Amino-ethyl)-benzonitrile hydrochloride (1.82 g, 10 mmol) inDMF (50 mL). After stirring at room temperature for 10 minutes,4-(4,6-Dimethoxy-[1,3,5]triazin-2-yl)-4-methyl-morpholin-4-ium chloride(DMTMM) (10 mmol) is added and the reaction stirred overnight at roomtemperature. The reaction mixture is partitioned between water (500 mL)and ethyl acetate (300 mL) and the aqueous layer is extracted with moreethyl acetate (100 mL). Combined ethyl acetate extracts is washed withsaturated NaHCO₃ (100 mL), and brine (100 mL). The organic layer isdried (Na₂SO₄), filtered and then evaporated under reduced pressure togive 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid[(R)-1-(3-cyano-phenyl)-ethyl]-amide as the major product (3.2 g) whichis taken directly into the next reaction (amidoxime formation).

Step 5

Hydroxylamine hydrochloride (1.52 g, 24.2 mmol) is added to a cooledsolution of 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid[(R)-1-(3-cyano-phenyl)-ethyl]-amide (3.2 g, 9.7 mmol) in methanol (40mL) and the suspension cooled in an ice water bath. Triethyl amine (2.44g, 24.2 mmol) is added to the flask and the reaction mixture is allowedto warm to room temperature over night. The reaction mixture evaporatedunder reduced pressure and the crude partitioned between water and ethylacetate. Organic layer is separated, dried (Na₂SO₄) and evaporated underreduced pressure. Toluene (50 mL) and CHCl₃ (50 mL) are added andevaporated under reduced pressure to give2-Pyridin-2-yl-pyrimidine-5-carboxylic acid{(R)-1-[3-(N-hydroxycarbamimidoyl)-phenyl]-ethyl}-amide (3 g) as themajor product.

MS: 363 (M+H).

¹H NMR (300 MHz, DMSO): δ=1.54 (d, 3H), 5.18-5.27 (m, 1H), 5.80 (bs,2H), 7.35 (t, 1H), 7.44 (d, 1H), 7.54-7.60 (m, 2H), 7.74 (s, 1H), 8.45(d, 1H), 8.79 (d, 1H), 9.26 (d, 1H), 9.35 (s, 2H), 9.60 (s, 1H).

Step 6 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amide

Methyl 2-hydroxy-2-methyl-propionate (2 mL) and K₂CO₃ (219 mg, 1.59mmol) are added to a microwave vial containing2-Pyridin-2-yl-pyrimidine-5-carboxylic acid{(R)-1-[3-(N-hydroxycarbamimidoyl)-phenyl]-ethyl}-amide (0.5 g, 1.38mmol) and heated at 180° C. in a microwave for 10 minutes. The reactionmixture is evaporated under reduced pressure and purified by reversephase HPLC to give 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amideas the major compound (110 mg).

MS: 431 (M+H).

¹H NMR (300 MHz, DMSO): δ=1.58 (d, 3H), 1.61 (s, 6H), 5.27-5.31 (m, 1H),6.08 (s, 1H), 7.53-7.60 (m, 2H), 7.67 (d, 1H), 7.91 (d, 1H), 8.02 (t,1H), 8.08 (s, 1H), 8.45 (d, 1H), 8.79 (d, 1H), 9.35-9.39 (m, 3H).

2-Pyridin-2-yl-pyrimidine-5-carboxylic acid((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-ethyl)-amidecan also be prepared by following procedures similar to those of Example2 but substituting2-{3-[3-((R)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl}-propan-2-olHydrochloride for2-{3-[3-((S)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl}-propan-2-olHydrochloride.

In Vitro Assay Protocols to Identify Inhibitors of Hematopoietic PGD2Synthase

The compounds of the present invention can be tested for enzymaticinhibiting activity against PGD2 Synthase according to either one of thefollowing assays.

Assay 1: Fluorescence Polarization Assay

As described in PCT publication WO 2004/016223, Example II.

Assay 2: Enzyme Immunoassay (EIA) Method I. Assay Solutions

-   -   a. Preparation of 0.1M K₂HPO₄/KH₂PO₄ buffer (pH 7.4)        -   Prepare 0.1 M KH₂PO₄ from 1M KH₂PO₄ (Sigma, Cat# P-8709)        -   Prepare 0.1 M K₂HPO₄ from powder of K₂HPO₄ (Fisher,            BP363-500)        -   Mix 0.1 M K₂HPO₄ with 0.1 M KH₂PO₄ to adjust pH to 7.4.    -   b. Preparation of 0.5% γ-globulin        -   Add 0.1 g of γ-globulin (Sigma, Cat# G-5009) to 20 mL 0.1 M            K₂HPO₄/KH₂PO₄        -   buffer (pH 7.4) and make 1-mL/vial aliquots and store in            −80° C.    -   c. Preparation of 100 mM GSH        -   Add 307 mg of GSH (Sigma, Cat# G-6529) to 10 mL 0.1 M            K₂HPO₄/KH₂PO₄        -   buffer (pH 7.4) and store at −80° C.    -   d. Preparation of Reaction buffer:        -   198 mL of 0.1M K₂HPO₄/KH₂PO₄ buffer (pH 7.4)        -   2 mM GSH—Prepared from 100 mM GSH        -   0.4 g Glycerol        -   2 mL of 0.5% γ-globulin        -   Add 0.4 g of glycerol and 2 mL of 0.5% γ-globulin to 198 mL            of 0.1 M K₂HPO₄/KH₂PO₄        -   buffer (pH7.4).        -   Add 0.4 mL of 100 mM GSH to 19.6 mL reaction buffer before            the assay (enough for two 96-        -   well plates).    -   e. Preparation of FeCl₂/citric acid stopping solution: (8 mg/mL        FeCl₂, 0.1 M citric acid)        -   Add 40 mg fresh FeCl₂ (IGN, Cat#158046) to 5 mL 0.1 M citric            acid (Sigma, Cat#C0759).    -   f. Preparation of MOX reagent:        -   10% EtOH—Add 1 mL of EtOH to 9 mL of ultra pure H₂O        -   Dissolve 0.1 g of methoxylamine (Cayman, Cat#400036/) in 10%            EtOH (10 mL).        -   Add 0.82 g of sodium acetate (Cayman, Cat#400037) to MOX            solution and dissolve.

II. Materials and Method

-   -   Dimethylsulfoxide (DMSO; Sigma; Cat# D2650)    -   Prostaglandin D2-MOX express EIA kit (Caymen Chemical, Catalog        No. 500151)

Before the assay, cool down 10 mL of acetone in polypropylene tubes andempty 96 well plates in ice. All the procedures except compound dilutionare performed on ice.

III. Compound Dilution

-   -   1. Dilute compound in DMSO

Vol of DMSO stock solution Compound concentration (μL) DMSO (μL) (mM) 4μL of 10 mM 6 μL 4 3 μL of 4 mM 6 μL 1.3333 3 μL of 1.33 mM 6 μL 0.44443 μL of 0.44 mM 6 μL 0.1481 3 μL of 0.148 mM 6 μL 0.0494 3 μL of 0.049mM 6 μL 0.0165 3 μL of 0.016 mM 6 μL 0.0055

-   -   2. Dilute 2 μL of each above concentration of compound to 38 μL        of reaction buffer in 96-well plates and mix.

IV. Enzyme and Substrate Solution Preparation

-   -   1. Preparation of 0.39 ng/μL enzyme solution (0.35 ng/μL at        final after compound addition).        -   Mix 4 μL of 4 mg/mL human h-PGDS with 396 μL of reaction            buffer (to give enzyme concentration 40 μg/mL). Add 46.8 μL            of 40 μg/mL h-PGDS to 4.753 mL of reaction buffer to give a            total volume of 4.8 mL    -   2. Preparation of Substrate Solution (PGH2): Add 0.375 mL of 0.1        mg/mL of PGH2 to 1.625 mL acetone.

V. Enzyme Reaction:

-   -   1. Add 60 μL of enzyme solution to compound well and positive        control (without compound) in U-bottom polypropylene plate on        ice.    -   2. Add 60 μL of reaction buffer and 6.6 μL of 5% DMSO in        reaction buffer into negative control wells in the plate.    -   3. Add 6.6 μL of diluted compound in reaction buffer to the        compound wells and mix.    -   4. Add 6.6 μL of 5% DMSO in reaction buffer to the positive        control well.    -   5. Incubate the plate in ice for at least 30 min.    -   6. Add 20 μL of substrate (PGH2) solution to compound, negative        and positive control wells in the U-bottom 96 well plate on ice.    -   7. Dry the plate in cold room for about 25-28 min.    -   8. Pipette 45 μL of enzyme solution (above) into 96-wells with        dried PGH2 and mix 3 times. Incubate on the ice for 1 min.    -   9. Add 45 μL of FeCl₂ solution into each wells and mix.    -   10. Add 90 μL of MOX solution and mix.    -   11. Incubate for 30 min at 60° C.    -   12. Dilute the samples 2500× with EIA buffer.

VI. EIA Assay

-   -   Perform the assay according to the procedure in EIA kit provided        by Cayman. Total PGD2 levels (pg/mL) were determined in the        samples by EIA kits (Cayman Chemical, Catalog No. 500151)

Calculate amount of PGD2 as below

-   -   Calculated % Positive control according to the equation below;

% Positive control=(Compound value−Negative control)/(Positivevalue−Negative control value)×100.

${\% \mspace{14mu} {Positive}\mspace{14mu} {control}} = {\frac{\left( {{{Compound}\mspace{14mu} {value}} - {{Negative}\mspace{14mu} {control}}} \right)}{\left( {{{Positive}\mspace{14mu} {value}} - {{Negative}\mspace{14mu} {control}\mspace{14mu} {value}}} \right)} \times 100}$

-   -   Compound value=PGD2 levels (pg/mL) obtained from the standard        curve in EIA assay for the samples with compound    -   Negative control value=PGD2 levels (pg/mL) obtained from the        standard curve in EIA assay for the samples without enzyme    -   Positive control value=PGD2 levels (pg/mL) obtained from the        standard curve in EIA assay for the samples with enzyme but        without compound    -   IC₅₀s are determined by excel fit to get the x value when        y=½Ymax using 4 parameter logistic model for the IC₅₀ curves.

Results

Compounds within the scope of the invention produce 50% inhibition inthe Fluorescence Polarization Assay or the EIA assay at concentrationswithin the range of about 1 nanomolar to about 30 micromolar,particularly about 1 nanomolar to about 1 micromolar, and moreparticularly about 1 nanomolar to about 100 nanomolar.

hPGDS EIA IC50 Example nM solidsol μM 1 12 135.9 2 11 854.9 3 26 29.4

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof.

1. A compound of formula (I):

wherein R1 is hydrogen or C₁-C₆alkyl; R2 is hydrogen, halogen orC₁-C₃alkyl; and R3 is hydroxyalkyl; or a pharmaceutically acceptablesalt thereof. 2-24. (canceled)